Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
  • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
  • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
  • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
  • A61K47/6817—Toxins
  • A61K47/6819—Plant toxins
  • A61K47/6825—Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
  • A61K47/6817—Toxins
  • A61K47/6819—Plant toxins
  • A61K47/6825—Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
  • A61K47/6827—Ricin A
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
  • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
  • C07D233/56—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
  • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
  • C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to new cytotoxic conjugates, a process for their preparation and pharmaceutical compositions having a cytotoxic action.
• U.S. Pat. No. 4,340,535 describes the preparation of anticancer products, called conjugates or also immunotoxins, obtained by the coupling, by means of a covalent bond, of the A chain of ricin with a protein structure, such as an antibody, an immunoglobulin or a fragment of an immunoglobulin, which is capable of selectively recognizing a given antigen on the surface of the cells carrying this antigen, such as the cancerous cells, which it is desired to attack.
• conjugates are specific cytotoxic agents for the intended target cells.
• the conjugates possessing these properties were always artificial mixed molecules in which the A chain of ricin was associated, by means of a covalent bond of the disulfide type, with an antibody, an immunoglobulin or a fragment of an immunoglobulin capable of selectively recognizing an antigen carried by the intended target cells.
• the covalent bond between the A chain of ricin and the antibody contains a disulfide radical
• one of the sulfur atoms forming the disulfide bond is always the sulfur atom belonging to the cysteine residue in the 257-position of the A chain of ricin, and
• the bonding arm joining the A chain of ricin and the antibody is fixed to the latter at NH 2 side or end groups of a peptide chain.
• the present invention relates to products, belonging to the class of the immunotoxins, obtained by the covalent coupling of, oh the one hand, the A chain of ricin, as such or appropriately modified (denoted hereafter by the symbol A), with, on the other hand, an antibody or a fragment of an antibody, used in its natural form or correctly modified (denoted hereafter by the symbol P), which possesses the capacity to selectively recognize an antigen carried by the intended target cells.
• the coupling of the 2 proteins can be effected either via a disulfide bond or via a thioether bond.
• the present invention relates to an immunotoxin formed by the coupling of an antibody P with the sub-unit A of ricin, having the following statistical formula:
• P' represents the radical of a protein which is an antibody or a fragment of an antibody, as such or appropriately chemically modified, from which at least one of its own groups has been removed and in which the other functional groups are optionally blocked
• A' represents the radical of a protein which is the sub-unit A of ricin, as such or appropriately chemically modified, from which at least one of its own groups has been removed and in which the other functional groups are optionally blocked
• W represents a divalent covalent structure containing a thioether group or a disulfide group in which either the sulfur atoms are those of the cysteines of p and A or they are bonded to the groups belonging to P and/or A by spacing structures carrying a functional group bonded to the said groups belonging to P and/or A, with the limitation that, in the case where W contains a disulfide group, when one of the sulfur atoms of the said disulfide is that belonging to one of the cysteines of A, the other sulfur is bonded to the protein P by a spacing structure carrying
• a thioether bond between two proteins is understood as meaning a bond of the type: ##STR1## in which Z, Y and E are as defined below.
• the present invention relates preferentially to an immunotoxin of the statistical formula:
• Z and Z' represent the groups belonging to the proteins A and P, chosen from the oxygen atom originating from the hydroxyl of one of the tyrosine residues, the carbonyl group originating from one of the terminal carboxyls or the free carboxyls of the aspartic and/or glutamic acids of A and P, the group originating from the dialdehyde structure obtained after oxidation of the carbohydrate structure of P with periodic acid, and the --NH-- group originating from one of the terminal amines of A and P or from one of the amines in the epsilon position of one of the lysine residues;
• Z" is as defined above for Z and Z' but cannot be --NH--;
• Y and Y' represent functional groups capable of bonding covalently with any one of the groups Z, Z' and Z" of the proteins A and P;
• E and E' represent inert spacing structures
• n zero or 1.
• the immunotoxins of the present invention are represented in simplified form by the formulae I and II above, but it is understood that the divalent covalent structure --W-- or --W'-- is bonded to at least one molecule P and at least one molecule A.
• the number of bonds with the proteins P and A depends on the number of groups belonging to the said proteins which are involved in the coupling operation.
• an immunotoxin is formed by the coupling of the sub-unit A of native ricin with the antibody P (for example the antibody T101) via a divalent covalent structure having a disulfide group in which one sulfur is that belonging to the 257-cysteine of the A chain of ricin and the other is bonded to the phenolic oxygens of the tyrosines of the antibody P by an oxopropyl group, it will have the statistical formula:
• t represents the number of tyrosines in the antibody (for example the antibody T101) which are involved in the coupling.
• P' is as defined above, especially the radical of the antibody T101 from which t phenolic groups of its tyrosines have been removed;
• A' is as defined above, especially the radical of the A chain of ricin from which the thiol group of its 257-cysteine has been removed;
• W' is the group (c):
• Z" is the oxygen of the phenolic hydroxyls involved in the coupling
• Y is --CO--
• E is the inert spacing structure --CH 2 --CH 2 -- and n is zero.
• immunotoxins formed by one or more structures containing the sub-unit A of ricin and a single antibody P, which are represented by the statistical formula:
• m represents the number of groups belonging to the protein P which are involved in the coupling.
• the number m varies from 0.3 to 12, preferably from 0.5 to 10.
• m varies from 0.3 to 12, preferably from 0.5 to 10
• m varies from 0.3 to 12, preferably from 0.5 to 10
• the expression "m varies from 0.3 to 12, preferably from 0.5 to 10" means that the value of m is a statistical value because the coupling does not take place homogeneously within the population of antibody molecules.
• the number m may therefore not be an integer.
• m depends especially on the antibodies used and more particularly on their molecular weight.
• the value of m can vary between 0.3 and about 2; if a fragment F(ab') 2 is used, m can vary between 0.5 and about 4; for an antibody of the IgG type, the value of m will be between 0.5 and 6; finally, for an antibody IgM, the value of m can vary between 1 and 12.
• the degree of substitution on the antibody p is such as to lead to a value of m which is not less than 0.5 and not more than 10.
• the present invention relates to a process for the preparation of an immunotoxin having the structure I above, wherein a protein P 1 , which is arbitrarily either the optionally modified sub-unit A of ricin or an antibody or a fragment of an antibody, carrying at least one free thiol group attached to the said protein P 1 directly or via a spacing structure, is reacted, in aqueous solution and at ambient temperature, with a protein P 2 different from P 1 , which is arbitrarily either the sub-unit A of ricin or an antibody or a fragment of an antibody, carrying a group capable of coupling with the free thiol of the protein P 1 to form a thioether or disulfide bond, with the limitation that, in the- case of the formation of the disulfide bond, when the protein P 1 is the sub-unit A of ricin, the bond with the protein P 2 is formed with a group of the said protein P 2 other than the amine groups.
• the present invention relates to a process for the preparation of an immunotoxin having the structure II, in which P', W' and A' are as defined above, wherein a protein of the formula:
• P 1 ' and P 2 ' represent the radicals of the proteins P 1 and P 2 bonded to the groups belonging to the said proteins, or the radicals of one of the protein P 1 or P 2 originating from the opening of the carbohydrate structures of antibodies or antibody fragments by reaction with periodic acid
• Z, Z', Y, Y', E et E' are as defined above and G represents a group : ##STR4## or a group --S--S---X, in which X is an activating group, it being understood that, when G is a group --S--S--X and P 1 ' is the sub-unit A of ricin, n is 1 or alternatively n can be zero, but, in this case, Z' is other than --NH--.
• the said thiol groups and functional groups are those of the native proteins p or A or alternatively are introduced therein artificially.
• the symbol P represents a protein chosen from any antibody or fragment of an antibody, any immunoglobulin or fragment of an immunoglobulin, or any molecule derived from these molecules by artifical modification of any one of their functional groups, including carbohydrate structures which they carry, with the proviso that the chosen protein is still capable of selectively recognizing a given antigen on the surface of the cells carrying this antigen, especially cancerous cells.
• the symbol A represents a protein which is the sub-unit, called the A chain, of the plant toxin ricin, such as can be obtained directly from natural ricin, or any molecule derived from this A chain by artificial modification of any functional group carried by this protein with the proviso that the chosen protein still has the property of inhibiting ribosomal protein synthesis in the eucaryotic cells, as can be demonstrated in an acellular study model.
• the symbol P' represents a radical derived from the above protein P, as such or appropriately chemically modified, from which one or more of its own groups have been removed and in which other functional groups are optionally blocked.
• A' represents a radical derived from the above protein A, as such or appropriately chemically modified, from which one or more of its own groups have been removed and in which other functional groups are optionally blocked.
• P 1 represents one of the proteins A and P as defined above, which carries free thiol groups attached to the said protein directly or via a spacing structure.
• P 2 which is different from P 1 , represents one of the proteins A and P as defined above, which carries one or more functional groups capable of reacting with the free thiols.
• the symbol P 1 ' represents that radical of the protein P 1 which is bonded to the groups belonging to the protein P 1 , especially the groups SH (of the cysteine), NH 2 (in the terminal position of the protein or in the epsilon position of the lysines), OH (of the tyrosines) or COOH (of the aspartic and glutamic acids), or that radical of the protein P 1 which originates from the opening, by reaction with periodic acid, of the carbohydrate structures, when P 1 is an antibody or an antibody fragment.
• SH of the cysteine
• NH 2 in the terminal position of the protein or in the epsilon position of the lysines
• OH of the tyrosines
• COOH of the aspartic and glutamic acids
• the symbol P 2 ' represents that radical of the protein P 2 which is bonded to the characteristiccritical groups NH 2 (in the terminal position of the protein or in the epsilon position of the lysines), OH (of the tyrosines) or COOH (of the aspartic and glutamic acids).
• P 1 '--SH represents the protein P 1 (which can arbitrarily be the antibody or antibody fragment P or the sub-unit A of ricin) in which the SH groups of the cysteines are free and the other functional groups are optionally blocked.
• P 1 '--CO-- represents the protein P 1 which the terminal carboxyl group or the carboxyl groups of its glutamic and aspartic acids are coupled with a group which artificially introduces an SH group.
• P 2 '--NH-- represents the protein P 2 (which can arbitrarily be the antibody or antibody fragment P or the sub-unit A of ricin) in which the terminal amino group or the amino groups of its lysines are attached to a group capable of coupling with the thiol of the protein P 1 .
• inert spacing structure denotes a divalent organic radical which is inert towards the reactants used in the process, such as a straight-chain or branched alkylene group containing from 1 to 15 carbon atoms, which can contain one or more double bonds, can be interrupted by oxygen atoms or can be substituted by one or more inert functional groups such as methoxy groups, free or esterified carboxyl groups, dialkylamino groups or carbamate groups.
• inert functional groups such as methoxy groups, free or esterified carboxyl groups, dialkylamino groups or carbamate groups.
• the same term also denotes an arylene group containing from 6 to 15 carbon atoms, which can be substituted by one or more inert functional groups as indicated above for the alkylene group.
• the groups --CO-- and --(C ⁇ NH)-- are suitable functional groups capable of bonding with the free amines, the thiols and the phenolic hydroxyls of the proteins.
• the --NH-- group is a suitable functional group capable of bonding with the free carboxyl groups of the proteins.
• the group ⁇ N-- is a suitable functional group capable of bonding with the two carbon atoms of the carbohydrate structures of the proteins P 1 or P 2 after oxidation with periodate ions, when P 1 or P 2 is an antibody or antibody fragment.
• group belonging to the proteins denotes the radicals originating from the characteristic groups of the amino acids forming the proteins P 1 and P 2 , such as the oxygen atom originating from the hydroxyls of the tyrosine and possibly serine amino acids, the carbonyl group originating from the terminal carboxyl or the free carboxyls of the aspartic and glutamic acids, the --NH-- group originating from the terminal amine of the proteins or the lysines, or the sulfur atom originating from the thiol of the cysteine.
• the same expression also denotes the group originating from the dialdehyde structure obtained after oxidation of one of the carbohydrate structures of the proteins P 1 or P 2 by treatment with periodate ions, when P 1 or P 2 is antibody or antibody fragment.
• activating radical denotes a group bonded to an --S--S-- bridge and capable of reacting with a free thiol to form a disulfide with the release of X-SH.
• Suitable activating radicals are pyridin-2-yl and pyridin-4-yl groups which are unsubstituted or substituted by one or more halogens or alkyl, carboxyl or alkoxycarbonyl radicals; the phenyl group which is unsubstituted or, preferably, substituted by one or more halogens or nitro, alkoxy, carboxyl or alkoxycarbonyl groups; or an alkoxycarbonyl group such as methoxycarbonyl.
• alkyl and “alkoxy” denote groups containing up to 5 carbon atoms.
• alkylene denotes straight-chain or branched, saturated aliphatic groups containing up to 10 carbon atoms, which can be substituted by one or more inert functional groups such as alkoxycarbonyl groups.
• thiol groups for forming the bond between the 2 proteins.
• these thiol groups are particularly suitable for forming either disulfide bonds or thioether bonds, both of which satisfy the general conditions above.
• the spacing structure E can be replaced by the preferred structures R to R 8 , which are only given as examples.
• the protein P 1 carries, in the natural state, one or more thiol radicals which can be used to permit coupling with the protein P 2 ; this is particularly the case if the protein P 1 is the antibody fragment known as F(ab)', as conventionally obtained by the limited proteolysis of the antibody in the presence of pepsin, followed by reduction of the disulfide bridge (or bridges) between high-molecular chains.
• the protein P 1 is the A chain of ricin or a derivative of this A chain in which at least one of the thiol groups carried by the 171-cysteine and 257-cysteine residues of the A chain of native ricin is free and accessible for chemical coupling.
• the protein P 1 carrying its natural thiol group (or groups) can be used in this state for the coupling step.
• the protein P 1 does not carry, in the natural state, thiol radicals which can be used to permit coupling with the protein P 2 ;
• the protein P 1 is a native immunoglobulin, a whole antibody or a fragment of an antibody, especially one of the fragments commonly called F(ab)' 2 or F(ab);
• the first type of reaction is with S-acetylmercaptosuccinic anhydride, which is capable of acylating amine groups of the protein. It will then be possible to free the thiol groups by reaction with hydroxylamine to remove the acetyl protecting radical, in the manner already described (Archives of Biochemistry and Biophysics, 119, 41-49, 1967).
• the second type of reaction consists in reacting the protein via its carboxyl groups with a symmetrical diamino molecule having a disulfide bridge, of the formula:
• R 1 is an aliphatic group containing from 2 to 5 carbon atoms.
• a coupling agent such as a carbodiimide and especially a water-soluble derivative like 1-ethyl-3-dimethylaminopropyl-3-carbodiimide
• reaction product of this type can then be used in two ways:
• the product thus obtained is then purified by dialysis or gel filtration.
• the radical P 1 ' is that radical of the protein P 1 which consists of an antibody or one of its fragments
• the reaction medium obtained will be used as such for the coupling, in which case a thiol/disulfide exchange method will be used, for example the one described by Gilliland and Collier (Cancer Research, 40, 3564, 1980).
• the third type of reaction consists in using carbohydrate units, which are present in the natural state in the antibodies, in order to fix the radical carrying the thiol which it is proposed to introduce.
• the protein is then subjected to oxidation with periodate ions in order to create aldehyde groups on the carbohydrate units.
• the product obtained is treated with a symmetrical diamino molecule having a disulfide bridge, of the general formula:
• R 1 is an aliphatic group containing from 2 to 5 carbon atoms.
• the addition products formed are then reduced to secondary or tertiary amines by reaction with a suitable metal hydride, especially sodium borohydride.
• a suitable metal hydride especially sodium borohydride.
• reaction medium obtained may then be treated exactly as indicated above for the products characterized by the structures Ia or Ib, wherein P' 1 is an antibody or an antibody fragment.
• the protein P 1 used preferably possesses neither free SH groups nor free amino groups.
• This protein is in all cases the one which carries one or more functional groups capable of reacting with the thiols of the protein P 1 to form either a disulfide or a thioether bond.
• These functional groups which are always introduced artificially into the protein P 2 , differ according to whether it is desired to effect coupling by a disulfide bond or by a thioether bond and are chosen as indicated below.
• the protein P 2 substituted by an activated sulfur atom is obtained from the protein P 2 itself or from the correctly protected protein P 2 by substitution with the aid of a reagent which itself carries an activated sulphur atom, according to the equation:
• P 2 denotes the protein to be substituted
• L--Y' represents a group permitting the covalent fixation of the reagent to the protein.
• the functional group L--Y' is a group capable of bonding covalently with any one of the groups carried by the side chains of the constituent amino acids of the protein to be substituted.
• these groups the following will be singled out in particular:
• L--Y' can represent especially:
• a carboxyl group which can bond to the amino groups of the protein in the presence of a coupling agent such as a carbodiimide and especially a water-soluble derivative like 1-ethyl-3-dimethylaminopropyl-3-carbodiimide;
• activated ester such as an ortho- or para-nitrophenyl or -dinitrophenyl ester, or alternatively an N-hydroxysuccinimide ester, which can react directly with the amino groups to acylate them;
• an internal anhydride of a dicarboxylic acid such as, for example, succinic anhydride, which reacts spontaneously with the amine groups to create amide bonds
• R 2 is an alkyl group, which reacts with the amino groups of the protein P 2 according to the equation: ##STR7## in which R 3 represents the group --R--S--SX;
• L--Y' can represent especially an imidazol-1-ylcarbonyl group, which reacts with the phenol groups of the protein according to the equation: ##STR8## in which the imidazol-1-yl is L, the CO group is Y' and R 4 is the group --R--S--S--X.
• the radical --S--S--X denotes an activated mixed disulfide capable of reacting with a free thiol radical.
• X can denote a pyridin-2-yl or pyridin-4-yl group optionally substituted by one or more alkyl, halogen or carboxyl radicals.
• X can also denote a phenyl group preferably substituted by one or more nitro or carboxyl groups.
• X can represent an alkoxycarbonyl group such as the methoxycarbonyl group.
• the radical R denotes the spacing structure (indicated as E in the general formula II above) capable of carrying the substituents Y' and S--S--X simultaneously. It must be chosen so as not to contain groups capable of interfering, during the subsequent reactions, with the reactants used and the products synthesized.
• the group R can be a group --(CH 2 ) n --, n being between 1 and 10, or alternatively a group: ##STR9## in which R 6 denotes hydrogen or an alkyl group having from 1 to 8 carbon atoms and R 5 denotes a substituent which is inert towards the reactants to be used subsequently, such as a carbamate group: ##STR10## in which R 7 denotes a linear or branched alkyl group having from 1 to 5 carbon atoms, especially the tert.-butyl group.
• the reaction of the compound L--Y'--R---S----X with the protein P 2 is carried out in a homogeneous liquid phase, most commonly in water or a buffer solution.
• a water-miscible organic solvent can be added to the reaction medium at a final concentration which can reach 20% by volume in the case of a tertiary alcohol, such as tertiary butanol, or 10% by volume in the case of dimethylformamide or tetrahydrofuran.
• the reaction is carried out at ambient temperature for a time varying from a few minutes to a few hours, after which the low molecular weight products, and in particular the excess reactants, can be removed by dialysis or gel filtration. This process usually makes it possible to introduce between 1 and 15 substituent groups per mol of protein.
• the coupling with the protein P 1 is carried out by bringing the two proteins together in an aqueous solution having a pH of between 6 and 8, at a temperature not exceeding 30° C., for a time varying from 1 hour to 24 hours.
• the aqueous solution obtained is dialyzed, if appropriate, to remove the low molecular weight products, and the conjugate can then be purified by a variety of known methods.
• the preparation of the conjugate consists in reacting P 1 '--(Z--Y--E) n --SH with the protein P 2 into which one or more maleimide radicals have been introduced beforehand.
• R 8 represents an aliphatic or aromatic spacing structure containing from 1 to 15 carbon atoms, which is inert towards the reactants to be used subsequently, and
• Z' represents groups which can vary according to the type of functional group of the protein P 2 involved in the coupling.
• Z' oxygen in the case of an ester on the phenol of a tyrosyl residue
• Z' NH in the case of the coupling of an activated carboxyl group with an amino group of the protein
• Z' NH--CH 2 in the case of the reaction of a chloromethyl ketone with an amino group of the protein.
• the protein P 2 substituted by the maleimide group or groups is obtained from the protein P 2 itself, or the correctly protected protein P 2 , by substitution of suitable groups of the protein with the aid of a reagent which itself carries the maleimide group.
• suitable groups the following will be singled out in particular:
• the reagent carrying the maleimide radical can be:
• a carboxyl group in which case the reaction is carried out, after activation of the carboxyl group, in the presence of a coupling agent such as a carbodiimide and especially a water-soluble derivative such as 1-ethyl-3-dimethylaminopropyl-3-carbodiimide,
• activated ester such as an ortho- or para-nitrophenyl or -dinitrophenyl ester, or alternatively an N-hydroxysuccinimide ester, which reacts directly with the amino groups to acylate them.
• the reagent carrying the maleimide radical can be a reagent of the general formula: ##STR15## which reacts with the phenol groups of the protein according to the equation: ##STR16##
• the reaction of the maleimide-carrying reagents with the protein P 2 is carried out in a homogeneous liquid phase, most commonly in water or a buffer solution. If necessitated by the solubility of the reactants, it is possible to add, to the reaction medium, a water-miscible organic solvent at a final concentration which can reach 20% by volume in the case of a tertiary alcohol, such as tertiary butanol, or 10% by volume in the case of dimethylformamide or tetrahydrofuran.
• the reaction is carried out at ambient temperature for a time varying from a few minutes to a few hours, after which the low molecular weight products, and in particular the excess reactants, can be removed by dialysis or gel filtration. This process usually makes it possible to introduce between 1 and 15 substituent groups per mol of protein.
• the coupling with the protein P 1 is carried out by bringing the two proteins together in an aqueous solution having a pH of between 6 and 8, at a temperature not exceeding 30° C., for a time varying from 1 hour to 24 hours.
• the solution obtained is dialyzed, if appropriate, to remove the low molecular weight products, and the conjugate can then be purified by a variety of known methods.
• those which are particularly suitable are the compounds of the formula II in which W' represents one of the groups (a), (b), (c) and (d) above, in which E and E' represent a group --(CH 2 ) p --, p being an integer from 2 to 7, or a group: ##STR17##
• the present invention relates to new products having the following statistical formula:
• P 2 " represents the radical of a protein chosen from:
• the oxygen atom is that belonging to the phenolic hydroxyl groups missing from the radical P 2 ";
• E represents a group --(CH 2 ) p --, in which p is an integer from 2 to 7, or a group: ##STR18## and G is a group of the structure --S--S--X, in which X is an activating radical chosen from the pyridin-2-yl and pyridin-4-yl groups which are unsubstituted or substituted by one or more halogens or alkyl, carboxyl or alkoxycarbonyl radicals, the phenyl group which is unsubstituted or substituted by one or more halogens or nitro, alkoxy, carboxyl or alkoxycarbonyl groups, or an alkoxycarbonyl group.
• the products of the formula VI are prepared by reacting a product of the formula:
• the antibodies used in these examples are:
• T101 directed against the antigen T65 present on human T lymphocytes and numerous lines of human T leukemia cells.
• This antibody is the one described in Journal of immunology 125(2), 725-7 (1980). It is commercially available from HYBRITECH Inc., SAN DIEGO, Calif., USA.
• P 1 A chain of ricin carrying an SH introduced via the carboxyls.
• P 2 antibody T101 into which an activated disulfide group has been introduced via the amines.
• the disulfide bridge of the cystamine fixed to the protein is then reduced with 2-mercaptoethanol (at a final concentration of 3 percent for 1 hour at 30° C.). Dialysis against the 125 mM phosphate buffer of pH 7 is then continued as before. After centrifugation, 14 ml of solution of modified A chain (NEM) containing 1.75 mg/ml are obtained. 0.7 free SH group per mol of protein can be determined in this product by ELLMAN's method. When examined by polyacrylamide gradient electrophoresis, this modified A chain (NEM) gives a single band with a molecuIar weight of approx. 30,000.
• A' is the radical of the sub-unit A of ricin in which the SH groups are blocked with N-ethylmaleimide;
• P' is the radical of the antibody T101.
• W' is a group of the formula:
• Y' is --CO--
• E' is --CH 2 --CH 2 --
• E is --CH 2 --CH 2 --
• Y is --NH--
• n 1.
• the fundamental biological property of the A chain of ricin is to inhibit protein synthesis in eucaryotic cells by degradation of the ribosomal sub-unit 60S.
• test no. 1 The tests performed are therefore tests for the inhibition of protein synthesis: either on an acellular model (test no. 1) or on a cell model (test no. 2).
• test no. 1 The acellular model (test no. 1)
• the in vitro protocol uses appropriately complemented, subcellular fractions of rat liver capable of incorporating 14 C-phenylalanine in the presence of an artificial messenger RNA: polyuridylic acid.
• the procedure employed for preparing the subcellular fractions and measuring the incorporation of 14 C-phenylalanine is an adaptation of the method described in Biochemica Biophysica Acta 312, 608-615 (1973), using both a microsomal fraction and a cytosol fraction of the rat hepatocytes.
• the sample containing the A chain is introduced in the form of a solution appropriately diluted in a 50 mM Tris HCl buffer of pH 7.6, containing 0.2% of 2-mercaptoethanol and 15 micrograms/ml of bovine serum albumin.
• the count data are used to calculate, relative to a control medium without inhibitor, the percentage inhibition of the incorporation of 14 C-phenylalanine into the proteins for each reaction medium containing A chain of ricin. These values together make it possible to determine the concentration of A chain of ricin (or IC 50 ) which inhibits the incorporation of the 14 C-phenylalanine by 50% under the experimental conditions.
• This test measures the effect of the substances studied on the incorporation of 14 C-leucine into cancerous cells in culture.
• the cells used depend on the specificity of the antibody chosen to manufacture the immunotoxin. In this example, they are cells of the CEM human lymphoblastoid line which naturally carry the antigen T65.
• a--Test no. 1 (acellular model)
• the inhibitory activity of the modified A chain was determined. An IC 50 of 2.18.10 -10 mol/l is observed. The IC 50 of the control A chain in the experiment is 1.03.10 -10 mol/l; therefore, the modification does not cause a significant loss of activity of the A chain.
• the test is performed on CEM cells which naturally carry the antigen T65.
• the cytotoxic activity is thus 25,000 times higher than that of the A chain.
• P 1 A chain of ricin carrying an SH introduced via the carboxyls.
• P 2 antibody T101 into which a maleimide group has been introduced via the amines.
• A' is the radical of the sub-unit A of ricin in which the SH groups are blocked with N-ethylmaleimide;
• P' is the radical of the antibody T101.
• W' is a group of the formula: ##STR20## in which: Z is --NH--
• E is --(CH 2 ) 5 --
• E' is --(CH 2 ) 2 --
• Y' is --NH--
• Z' is --CO--
• n 1.
• a--Test no. 1 (acellular model)
• P 1 A chain of ricin into which an SH group has been introduced via the amines.
• P 2 antibody T101 into which an activated disulfide group has been introduced via the amines.
• the reaction medium is purified on a Sephadex G100 column in the manner described in Example 1 to give 24 ml of an immunotoxin solution containing 0.71 mg/ml, i.e. 17 mg.
• This solution contains 0.130 mg of modified A chain (NEM) coupled with the antibody.
• the average degree of coupling in this preparation is 1.1 modified A chains (NEM) per mol of antibody.
• A' is the radical of the sub-unit A of ricin in which the SH groups are blocked with N-ethylmaleimide;
• P' is the radical of the antibody T101.
• W' is a group of formula:
• n 1.
• the inhibitory activity of the modified A chaIn was determined: the IC 50 is equal to 1.95.10 -10 mol/l.
• the IC 50 of the control A chain is 1.5.10 -10 mol/l in the experiment.
• the modification does not cause a significant loss of activity of the A chain.
• the IC 50 of this immunotoxin is 10 -13 mol/l.
• P 1 A chain of ricin into which an SH group is introduced via the amines.
• P 2 antibody T101 into which a maleimide group is introduced via the amines.
• the reaction medium is purified on a Sephadex G100 column in the manner described- in Example 1. This gives 30 ml of an immunotoxin solution containing 0.51 mg/ml (i.e. 15.2 mg). This solution contains 0.08 mg of modified A chain (NEM) coupled with the antibody. The average degree of coupling in this preparation is 1.0 modified A chain (NEM) per mol of antibody.
• A' is the radical of the sub-unit A of ricin in which the SH groups are blocked with N-ethylmaleimide;
• P' is the radical of the antibody T101.
• W' is a group of formula: ##STR22## wherein Z is --NH--
• n 1.
• the IC 50 in the presence of activator is 3.3.10 -12 mol/l, which represents a cytotoxic activity 5,000 times greater than that of the A chain in the same experiment.
• P 1 antibody anti-DNP into which an SH group has been introduced via the amines.
• P 2 A chain of ricin into which a maleimide group has been introduced via the amines.
• the coupling reagent is prepared by condensing maleimidocaproic acid with 2-phenyl-1-aminoethyl chloromethyl ketone. ##STR24##
• the maleimidocaproic acid (1 equivalent) is dissolved in tetrahydrofuran (THF) in the presence of methyl chloroformate (1.1 equivalents) and N-ethylmorpholine (1.1 equivalents).
• the organic phase is extracted twice with water, dried over MgSO 4 and evaporated to dryness.
• the non-crystalline product obtained is identified by its NMR spectrum.
• the solution is purified to remove the excess reagent by dialysis against 125 mM phosphate buffer of pH 7, and centrifuged.
• NEM A chain
• the reaction medium is stirred for 2 hours at 4° C. and the solution is then purified to remove the excess reagents by dialysis against 125 mM phosphate buffer of pH 7 (20 hours at 400 ml/h). This gives 4.1 ml of a solution containing 8.6 mg/ml. After reaction with hydroxylamine, 3.5 SH groups per mol of antibody were determined by ELLMAN's method.
• A' is the radical of the sub-unit A of ricin in which the thiol groups are blocked with N-ethylmaleimide;
• P' is the radical of the antibody anti-DNP
• W' is a group of formula: ##STR25## in which: Z' is --NH--
• Y' is --CO--
• E' is ##STR26##
• E is ##STR27##
• Y is --CO--CO--CH 2 -- Z is --NH--
• n 1.
• the inhibitory activity of the modified A chain was determined.
• the IC 50 is equal to 0.25.10 -10 mol/liter, the IC 50 of the control A chain being 0.78.10 -10 mol/liter in the experiment.
• the modification therefore causes no loss of activity of the A chain.
• test is performed under operating conditions identical to those of Example 1, but using CEM cells labeled with TNP in accordance with the technique described by the Applicant Company in French Patent No. 78/27838, relating to the labeling of HeLa cells with TNP.
• the IC 50 of the immunotoxin in the presence of activator (10 mM NH 4 Cl) is 10 -8 mol/liter. It is 10 times lower than that found for the A chain.
• P 1 antibody T101 into which an SH has been introduced via the amines.
• P 2 A chain of ricin into which an activated disulfide group has been introduced via the hydroxyl of the tyrosines.
• the coupling reagent is the imidazolide derived from 3-(pyridin-2-yldisulfanyl)propionic acid (PDPA). This imidazolide is obtained in a single step from PDPA and carbonyldiimidazole (CDI). ##STR28##
• SAMSA S-acetylmercaptosuccinic anhydride
• antibody T101 containing 9 mg/ml, i.e. 0.3 micromol.
• the reaction medium is stirred for 2 hours at 4° C. and then purified to remove the reagents by dialysis against 125 mM phosphate buffer of pH 7 for 24 hours at a rate of 400 ml/hour. This gives 4.1 ml of a solution of modified antibody containing 8.6 mg/ml. 0.46 ml of a 0.5 M solution of hydroxylamine hydrochloride is added to this antibody solution. After incubation for 1 h at 30° C., the reaction medium is purified to remove the reagents by dialysis against 125 mM phosphate buffer of pH 7.
• SAMSA S-acetylmercaptosuccinic anhydride
• the reaction medium is purified on a Sephadex G100 column in the manner indicated in Example 1. This gives 17 ml of an immunotoxin solution containing 1.1 mg/ml (i.e. 18.7 mg). This solution contains 0.32 mg/ml of modified A chain (NEM). The average degree of coupling of this preparation is 2 A chains (NEM) per mol of antibody.
• A' is the radical of the sub-unit A of ricin in which the SH groups are blocked with N-ethylmaleimide;
• P' is the radical of the antibody T101.
• W' is a group of the formula:
• Y' is --CO--
• E' is ##STR29## E is --CH 2 CH 2 -- Y is --CO--
• n 1.
• the inhibitory activity of the modified A chain was determined.
• the IC 50 is equal to 3.6.10 -10 mol/liter.
• the IC 50 of the controI A chain is 1.2.10 -10 mol in the experiment. There is therefore no significant loss of activity of the modified A chain.
• P 1 antibody T101 into which an SH group has been introduced via the amines.
• P 2 A chain of ricin into which a maleimide group has been introduced via the hydroxyl of the tyrosines.
• the coupling agent is the imidazolide derived from maleimidocaproic acid. This imidazolide is obtained in a single step from maleimidocaproic acid and carbonyldiimidazole (CDI): ##STR30##
• the mixture is incubated for 1 h at 30° C.
• the residual maleimide groups are blocked with 5 micromol of cysteine. Incubation takes 1 hour at 30° C.
• the reaction mixture is purified on Sephadex G100 by the method described above. This gives 19.5 ml of an immunotoxin solution containing 1.05 mg/ml (i.e. 20.5 mg).
• This solution contains 0.31 mg/ml of modified A chain (NEM).
• the average degree of coupling of this preparation is 2 A chains (NEM) per mol of antibody.
• A' is the radical of the sub-unit A of ricin in which the SH groups are blocked with N-ethylmaleimide;
• P' is the radical of the antibody T101.
• W' is a group of the formula: ##STR31## in which: Z' is --NH--
• Y' is --CO--
• E' is ##STR32##
• E is --(CH 2 ) 5 -- Y is --CO--
• n 1.
• the inhibitory activity of the modified A chain was determined.
• the IC 50 is equal to 10.10 -10 mol/l.
• the IC 50 of the control A chain is 1.1.10 -10 mol/l in the experiment.
• the modified A chain still retains a high capacity to inhibit protein synthesis.
• P 1 A chain of ricin in the native state.
• P 2 antibody AT15E into which a maleimide group has been introduced via the amines.
• the antibody AT15E is modified by the method described in Example 2. By determination with 14 C-cysteine, it is found that 2.4 maleimide groups have been introduced per mol of antibody.
• A' is the radical of the sub-unit A of ricin
• P' is the radical of the antibody AT15E
• W' is a group of the formula: ##STR33## in which: Z is --NH--
• E is --(CH 2 ) 5 --
• n zero;
• m 1.8.
• the test is performed under operating conditions identical to those of Example 1, but using T murine leukemia cells carrying the antigen Thy 1.2.
• P 1 A chain of ricin in the native state.
• P 2 antibody T101 into which an activated disulfide group has been introduced via the hydroxyl of the tyrosines.
• A' is the radical of the sub-unit A of ricin
• P' is the radical of the antibody T101
• W' is a group of the formulae:
• Z" is --O--
• E is --CH 2 --CH 2 --
• n zero;
• m 1.5.
• P 1 A chain of ricin in the native state.
• P 2 antibody T101 into which a maleimide group has been introduced via the hydroxyl of the tyrosines.
• A' is the radical of the sub-unit A of ricin
• P' is the radical of the antibody T101
• W' is a group of the formula: ##STR34## in which: Z is --O--
• E is --(CH 2 ) 5 --
• n zero;
• the IC 50 of the immunotoxin in the presence of activator is 1.7.10 -12 mol/l, which represents an activity 3.10 3 times greater than that of the A chain in the same experiment.

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